Biochemistry 1986,25, 4784-4790
4784
Ridley, W. P., Houchins, J. P., & Kirkwood, S.(1975)J. Biol. Chem. 250, 8761-8767. Sutton, R., & Ferguson, S.J. (1985)Eur. J . Biochem. 148, 55 1-554. Tarr, G. E., Beecher, J. F., Bell, M., & McKean, D. J. (1978) Anal. Biochem. 84, 622-627.
Winge, D. R., Nielson, K. B., Zeikus, R. D., & Gray, W. R. (1984)J. Biol. Chem. 259, 11419-1 1425. Wolf, R. E., Jr., & Loper, J. C. (1969) J. Biol. Chem. 244, 6297-6303. Yourno, J. (1968)J. Biol. Chem. 243, 3277-3288. Yourno, J., & Ino, I. (1968)J. Biol. Chem. 243, 3273-3276.
Leaving Group Dependence in the Phosphorylation of Escherichia coli Alkaline Phosphatase by Monophosphate Esterst Adrian D. Hall and Andrew Williams* University Chemical Laboratories, Canterbury, Kent CT2 7NH, England Received January 31, 1986; Revised Manuscript Received April 3, 1986
ABSTRACT: Values of k,, and K , have been measured for the Escherichia coli alkaline phosphatase catalyzed hydrolysis of 18 aryl and 12 alkyl monophosphate esters at pH 8.00 and 25 "C. A Bransted plot of log (kcat/Km)(M-I s-') vs. the pK of the leaving hydroxyl group exhibits two regression lines:
+ 8.14 (f0.15) = -0.19 (*0.01) p p o H + 5.89 (f0.17)
log (kcat/Km)= -0.19 (f0.02)pKmH log ( k J K m )
Alkyl phosphates with aryl or large lipophilic side chains are not correlated by the above equations and occupy positions intermediate between the two lines. The observed change in effective charge on the leaving oxygen of the ester (-0.2) is very small, consistent with substantial electrophilic participation of the enzyme with this atom. Cyclohexylammonium ion is a noncompetitive inhibitor against 4-nitrophenyl phosphate substrate at p H 8.00,and neutral phenol is a competitive inhibitor (Ki = 82.6 mM); these data and the 100-fold larger reactivity of aryl over alkyl esters are consistent with the existence of a lipophilic binding site for the leaving group of the substrate. The absence of a major steric effect in k,,/K, for substituted aryl esters confirms that the leaving group in the enzymesubstrate complex points away from the surface of the enzyme. Arguments are advanced to exclude a dissociative mechanism (involving a metaphosphate ion) for the enzyme-catalyzed substitution at phosphorus.
x e presently accepted kinetic scheme for the catalytic action of alkaline phosphatase from Escherichia coli possesses four sequential steps passing through a central, phosphoryl-enzyme (eq 1). The phosphoryl group (-Pi = -P032-) becomes co-
the charge change may be defined. The most appropriate calibrating equilibrium is the hydrolysis of the monophosphate dianion (eq 2) for which the polar effect has recently been X-0-Pi
k4
E-HOPi EE k4
+ HOPi
(1)
valently attached to serine- 102 in the single peptide chain constituting a monomer of the dimeric enzyme (Coleman & Chlebowski, 1979;Bradshaw et al., 1981;Sowadski et al., 1985; Coleman & Gettins, 1983). There is now considerable information concerning the constituents of the active site from X-ray crystallographic, NMR spectroscopic, and chemical work providing ground-state spacial data. Knowledge of the change in charge on the leaving atom in transfer of the phosphoryl group from the substrate to the enzyme would provide useful data to describe the molecular mechanism of the catalysis. Measurement of the change in charge requires knowledge of the polar effect on the transition state for the phosphorylation step as well as a calibrating polar effect on an equilibrium for phosphoryl group transfer where 'This work was supported, in part, by the SERC (U.K.) (Grant GRC 75529).
X-0-H
+ H-O-Pi & X-0- + H+ X-0-H
(2) (3)
determined (Bourne & Williams, 1984a). All measurements of charge are referred to the ionization of the hydroxyl species (eq 3) where the charge change is defined as unity (Williams, 1984a). The rate-limiting step for the enzyme-catalyzed hydrolysis at low substrate concentrations is phosphorylation when the added leaving group concentration is negligible (k-,[XOH]
